Calcium (Ca2+) is a key regulator of a broad range of biological functions and is also a key element in the composition of dental enamel. Ca2+ must reach the forming enamel layer but how this process is regulated in ameloblasts is poorly understood. Our goal is to identify key pathways used by ameloblasts to regulate Ca2+ dynamics, in particular the required mechanisms for Ca2+ entry and pathways activated upon increases in cytosolic Ca2+ concentration. Deficiencies in the normal functioning of these pathways result in abnormal enamel that is prone to dental disease which can act as a host environment for oral bacteria. The focus of this proposal is to identify the functions of the store operated Ca2+ release-activated Ca2+ (CRAC) channels in enamel development. CRAC channels comprise important Ca2+ influx mechanisms activated following Ca2+ release from the endoplasmic reticulum (ER). The importance of Ca2+ influx via CRAC channels pathway in ameloblasts is understood by clinical reports describing hypo-calcified amelogenesis imperfecta in patients with mutations to STIM1 and ORAI1. However our understanding of CRAC channels function in enamel is limited as Stim1-/- and Orai1-/- animals die around birth. To address this problem, we developed several conditional knockout mice that specifically analyze the function of CRAC channels with particular reference to dental enamel formation. The ensuing Ca2+ entry via CRAC activates the calcineurin-NFAT pathway, which up-regulates the regulator of calcineurin (RCAN1). Although we find that NFAT and RCAN1 are expressed in enamel cells, and that this pathway is active during enamel development, the functions of these genes in enamel development are unknown. We are particularly interested in RCAN1 as Down syndrome patients present with a host of enamel deficiencies including abnormal mineralization and thinner enamel. Down syndrome is one of the most common human genetic disorders (frequency is 1 in ~700 births) characterized by elevated levels of RCAN1 in several tissues. The cause of growth alterations in the enamel of Down syndrome patients remains unknown. Our proposed studies will increase our understanding of enamel development. Such knowledge will impact caries prevention and has broader implications in bone homeostasis/development and in the development of ectodermal organs as some of these pathways are shared. The proposed work will also lead to a better understanding of systemic effects of CRAC channel function.